Minimally Invasive and in Situ Capacitive Sensing of Cardiac Troponin I from Interstitial Fluid

Figures

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  1. Schematic overview of the MiCaP for the in situ detection and quantification of dermal cTnI. (a) cTnI, released from the heart during MI events, transitions from the bloodstream into the ISF through capillary endothelial cells. Upon insertion of the functionalized MiCaP into the dermal layer, microneedles capture cTnI from ISF, and the concentration is quantified via capacitance interrogation of the MiCaP using a capacitance reading circuit. (b) Schematic illustration of the MiCaP design, showing the microneedle array and the interdigitated electrode pattern. (c) Three-dimensional illustration of MiCaP inserted into skin tissue. (d) Schematic of a single microneedle modified with antibodies for specific cTnI detection in dermis. (e) Representative variation of the sensor’s output metric (ΔC/C%), demonstrating MiCaP’s response to cTnI.

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  2. Fabrication and characterization of the MiCaP. (a) SEM image of the fabricated MiCaP (scale bar: 1 mm). (b) SEM image of a single microneedle (scale bar: 100 μm); the inset shows a magnified view of the microneedle tip (scale bar: 60 μm). (c) Optical image of MiCaP showing the IDE design with five fingers, each covering a row of five microneedles (scale bar: 2 mm). (d) Optical images demonstrating the mechanical flexibility of the MiCaP. (e) Optical image showing the setup for electrical resistance measurement from the microneedle tip to the contact pad (i) and summary of resistance measurements from 16 different microneedles (ii). (f) Optical images of MiCaP with the base covered with PDMS (left) and a single microneedle highlighting the exposed area of a microneedle to ISF (scale bar: 300 μm) (right). (g) CV of three independently fabricated MiCaP sensors, demonstrating consistent electrochemical performance. (h) EIS results of three MiCaP sensors, showing reproducible capacitance profiles across the 0.1–1000 kHz frequency range. (i) Result of continuous capacitance measurement of MiCaP exposed to 0.1 × PBS for 1800 s.

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  3. Performance characterization of the MiCaP in skin penetration. (a) SEM image showing indents created in rat skin after administration by the MiCaP (scale bar: 1 mm). (b) A zoomed-in view of an indent (scale bar: 100 μm). (c) Optical images for MiCaP insertion profile. Shaved and cleaned untreated rat skin, serving as the control (i), rat skin after treatment with MiCaP for 15 min. The visible indentations indicate successful microneedle insertion into the skin (ii), trypan blue spots within the rat skin resulting from the insertion of MiCaP stained with trypan blue (iii). The stained regions confirm the effective penetration of the microneedles into the skin. (d) Confocal microscopy image showing a penetration depth of at least 0.7 mm into rat skin. (e) SEM images of the MiCaP after skin penetration experiment (scale bar: 2 mm).

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  4. Electrical characterization of MiCaP. (a) Changes in EDL thickness at different stages of MiCaP functionalization and cTnI detection; in the bare sensor the EDL thickness is d 1 (i), then the sensor is functionalized with antibody and the thickness of the EDL is d 1 + d 2 (ii), after binding of cTnI molecules to the antibody the thickness of the EDL becomes d 1 + d 2 + d 3 (iii). (b) The equivalent circuit model of MiCaP when inserted in rat skin. (c) Measured impedance profile of the MiCaP (inserted in rat skin), used for curve fitting and circuit parameters extraction. (d) Plot of measured and fitted impedance data of the MiCaP for the frequency range of 1 to 100 kHz, indicating the accuracy of the fitting. (e) The simplified two-element equivalent circuit model of the MiCaP. (f) Measured and calculated C EDL for the frequency range of 1 to 100 kHz.

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  5. MiCaP biosensors development and characterization. (a) AFM images of the MiCaP before (i), and after antibody immobilization (ii). (b) EIS results indicate an increase in the R ct due to the absorption of anti-cTnI and MCH to MiCaP and the binding of 100 pg/mL of cTnI to the antibody. (c) MiCaP capacitance interrogation signal amplitude selection. For low amplitudes (<30 mV) of the excitation signal, the measured capacitance fluctuates and is not reliable. The capacitance became stable by increasing the signal to 30 and 50 mV. However, for higher signal levels (>100 mV), the applied signal triggers the EDL and leads to unreliable capacitance change in the sensor output. (d) Dose–response of the MiCaP for various concentrations of cTnI. (e) Calibration curve and calibration equation of the MiCaP. Each dose was repeated at least three times on a new functionalized sensor. Error bars indicate standard deviation. (f) Selectivity experiment of the MiCaP concerning seven different interfering molecules spiked in the buffer. Data were expressed as the mean ± SD; *P < 0.05, **P < 0.005, ***P < 0.001.

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  6. In vivo evaluation of MiCaP for cTnI detection in rat model. (a) Schematic overview of the in vivo experimental procedure. (b) Photograph of the experimental setup showing MiCaP attachment to the rat dorsal skin and real-time capacitance monitoring using the impedance analyzer. (c) MiCaP response (%ΔC/C) during in vivo testing in an experimental animal following cTnI administration. (d) MiCaP response (%ΔC/C) during in vivo testing in an control animal injected with blank physiological saline solution. (e) Summary of %ΔC/C values obtained from six rats (three experimental and three control animals). (f) Comparison of concentrations of cTnI measured in serum and ISF in the control group (left) and the experimental group (right) (n = 3 per group). cTnI in dermal ISF determined by MiCaP (green) exhibited a good qualitative correlation with that in serum tested by DXI 600 (red). (g) Summary of ISF and serum cTnI concentrations measured by MiCaP and DXI 600 across all six rats. Data are presented as mean ± SD. Statistical significance determined at **P < 0.005.